Bypassing mutant p53 in radiation therapy

在放射治疗中绕过突变型 p53

• 批准号: 8867861
• 负责人: Hongdau Peter Liu
• 金额: $ 4.81万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2017-06-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8867861
• 关键词: Aftercare; Apoptosis; Apoptotic; Biological Markers; Blinded; Bypass; Cancer Patient; Cancerous; Cell Culture Techniques; Cell Cycle Arrest; Cell Death; Cells; Cleaved cell; Clinical; DNA; DNA Damage; Embryo; FDA approved; Fishes; Genetic; Genetic Determinism; Genetic study; Hela Cells; Human; Ionizing radiation; Libraries; Malignant Neoplasms; Mediating; Methods; Modeling; Mutate; Mutation; PTEN gene; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Preclinical Drug Evaluation; Process; Radiation; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Radioresistance; Radiosensitization; Regulation; Research; Resistance; Signal Transduction; Specificity; Stratification; TP53 gene; Testing; Therapeutic; Tumor Suppressor Proteins; Zebrafish; base; cancer cell; cancer therapy; caspase-2; chemical genetics; chemotherapy; clinically relevant; improved; in vivo; inhibitor/antagonist; irradiation; mutant; neoplastic cell; novel; novel therapeutic intervention; oncology; protein complex; public health relevance; radiosensitizing; resistance mechanism; response; screening; small molecule; small molecule libraries; therapeutic target; tumor

Project Summary Mutation of the tumor suppressor TP53 leads to the evasion of apoptosis after radiation, underlying resistance to conventional cancer treatments, such as radiation and chemotherapy. Our research seeks to bypass these alterations through the discovery of parallel cell-death pathways. We previously discovered a novel apoptotic process, termed "Chk1-suppressed" (CS) pathway, which restores radiosensitivity in TP53 mutant cells after treatment with Chk1 inhibitors (Sidi et al. Cell 2008)1. Using both zebrafish and mammalian TP53-mutant models, our lab has established that the CS pathway requires the assembly of a protein complex called the PIDDosome, composed of PIDD, RAIDD, and caspase-2 (Ando et al. Mol Cell 2012)2. This proposal focuses on (1) identifying further genetic determinants predicting Chk1 inhibitor efficacy in radiotherapy and (2) discovering novel radiosensitizers against tumors with TP53 mutation. Utilizing both human cancer cells and zebrafish, we investigated the impact of cancer alterations other than mutant p53 on cellular sensitivity to CS apoptosis. We found that a mutation in PTEN, the second most commonly mutated tumor suppressor, blocks the execution of apoptosis after ionizing radiation (IR) and Chk1 inhibition (Chk1i). Furthermore, we identified the PTEN-Akt-IAP signaling axis as a candidate pathway to promote resistance to CS apoptosis. The loss of PTEN appears to inhibit the CS pathway downstream of caspase-2 activation, as HeLa cells with impaired PTEN fail to engage in apoptosis, despite an unaffected ability to cleave caspase-2 following IR+Chk1i. Our hypothesis is that one or more IAPs act downstream of Akt to directly inhibit active caspase-2. I propose to test this hypothesis using a candidate knockdown approach to determine which IAP(s), if any, is responsible for caspase-2 inhibition. The findings will be extended in vivo through both chemical and genetic studies in zebrafish. To identify novel targeted strategies for restoring radiosensitivity in TP53 mutant tumors, we exploited the high-throughput drug screening capability of zebrafish to perform a blinded phenotypic radiosensitization screen of 640 FDA-approved compounds. A primary screen of drugs treated in combination with 15 Gy IR yielded 140 compounds capable of producing phenotypes comparable to Chk1i (i.e. >75% sick or dead fish 4 days post irradiation) in p53 mutant fish. Our secondary screen of irradiated vs non-irradiated embryos so far has identified 6 compounds that demonstrate specific radiosensitizing effects. The genetic specificity of these candidate radiosensitizers will be confirmed and prioritized by clinical relevance, proposed mechanism of action, and efficacy. The proposed targets, along with the mechanism(s) of cell death will be confirmed in cell culture and in vivo. The results of these findings may aid in rapidly benefiting cancer patients by proposing a new application for an already approved and available drug.

项目概要 肿瘤抑制因子 TP53 的突变导致放射后细胞凋亡的逃避，潜在的 对传统癌症治疗（例如放疗和化疗）的抵抗力。我们的研究旨在 通过发现平行的细胞死亡途径来绕过这些改变。我们之前发现了一个 新型细胞凋亡过程，称为“Chk1 抑制”(CS) 途径，可恢复 TP53 的放射敏感性 用 Chk1 抑制剂处理后的突变细胞（Sidi 等人 Cell 2008）1。同时使用斑马鱼和哺乳动物 TP53突变模型，我们的实验室已经确定CS途径需要蛋白质复合物的组装 称为 PIDDosome，由 PIDD、RAIDD 和 caspase-2 组成（Ando 等人，Mol Cell 2012）2。这个提议 重点关注 (1) 进一步确定预测 Chk1 抑制剂在放疗中疗效的遗传决定因素，以及 (2) 发现针对 TP53 突变肿瘤的新型放射增敏剂。 利用人类癌细胞和斑马鱼，我们研究了癌症改变对其他细胞的影响。 细胞对 CS 凋亡的敏感性高于突变型 p53。我们发现 PTEN 发生突变，这是第二大突变。 常见突变的肿瘤抑制因子，可阻止电离辐射 (IR) 和 Chk1 后细胞凋亡的执行 抑制（Chk1i）。此外，我们确定 PTEN-Akt-IAP 信号轴作为候选途径 促进对CS细胞凋亡的抵抗。 PTEN 的缺失似乎抑制了 CS 通路下游 caspase-2 激活，因为 PTEN 受损的 HeLa 细胞无法参与细胞凋亡，尽管未受影响 IR+Chk1i 之后裂解 caspase-2 的能力。我们的假设是一个或多个 IAP 在 Akt 下游起作用 直接抑制活性 caspase-2。我建议使用候选击倒方法来测试这个假设 确定哪个 IAP（如果有）负责 caspase-2 抑制。研究结果将在体内推广 通过斑马鱼的化学和遗传学研究。 为了确定恢复 TP53 突变肿瘤放射敏感性的新靶向策略，我们利用 斑马鱼执行盲法表型放射增敏的高通量药物筛选能力 筛选 640 种 FDA 批准的化合物。与 15 Gy IR 联合治疗的药物的初步筛选 产生了 140 种能够产生与 Chk1i 相当的表型的化合物（即 >75% 患病或死亡的鱼 4 p53 突变鱼中的辐射后天数）。到目前为止，我们对经过辐射和未经辐射的胚胎进行了二次筛选 已鉴定出 6 种具有特定放射增敏作用的化合物。这些基因的特异性 候选放射增敏剂将根据临床相关性、拟议的机制进行确认和优先排序 行动、功效。所提出的目标以及细胞死亡的机制将在细胞中得到证实 培养和体内。这些发现的结果可能会通过提出一种方法来帮助癌症患者迅速受益 已批准且可用的药物的新申请。